Detection Method Establishment And Preliminary Application Of EMCV LAMP And Real-time PCR

Porcine encephalomyocarditis is a zoonotic infectious disease of natural focus caused by Encephalomyocarditis virus (EMCV), with myocarditis, encephalitis, myocardial inflammation, sow reproductive disorders and other symptoms. It is popular all over the world, and cause serious economic losses, in our country, the serological survey also found the infection among large-scale pig farms. Since EMCV is one of the important disease endangering the healthy development of pig industry in our country, therefore, it is very necessary to carry out the diagnosis and prevention of this virus, and it also has some significance to public health.In this study, it is intended to deign and synthesis primers to the LAMP reaction according to EMCV3D gene, and initially established a LAMP reaction system with25.0ul total volume; According to this system, the amplification products can be seen visually with obvious positive white precipitate, get some color after fluorochrome adding in, and electrophoresis result shows obvious trapezoidal strips.According to the established LAMP amplification reaction system, the Tm values, reaction time, temperature and other conditions were optimized, ultimately the determined inner and outer primer concentration ratio was5:1(inner and outer primers concentrations are of50μM,10μM respectively), the best reaction temperature was64℃, the reaction time for60min to reach the best reaction; On the specificity and sensitivity experiments, it proved that this method could detect EMCV with good specificity and sensitivity, about one order of magnitude higher compared with the conventional PCR; After detecting100swine serum samples selected at random by LAMP and ELISA, the results showed that the accuracy of LAMP to ELISA test was97.0%.On the other hand, one EMCV Real-time PCR reaction system was also set up successfully with the primer and probe designed to3D conservative gene, it has a25.0ul total volume, and the primers and probes was optimized through different combination, finally, the most suitable selected for this system were:primer EMCV LZD-F1/R1and the Probe1.According to the established EMCV Real-time PCR detection method, Tm value and concentration of primers were optimized, finally, the most suitable Tm value was60℃, upstream and downstream primer concentration were both10pmol/μL, probe concentration was5pmol/μL; On the specificity and sensitivity experiments, it proved that this method could detect EMCV with good specificity and sensitivity, about one hundred higher compared with the conventional PCR; With this established method, we detected98swine serum samples random selected, and compared the result with ELISA method detection, the result shows that the Real-time PCR and ELISA test results coincidence rate can reach98.0%.In conclusion, we successfully established EMCV LAMP and Real-time PCR detection method, both have good specificity, sensitivity, after detecting the clinical samples, it showed that coincidence rate with the ELISA detection are higher, and can be used in the clinical samples detection and analysis. LAMP method is more suitable for the primary detection, as it is simple, rapid and don’t need many equipments; the Real-time PCR is more suitable for scientific experiment. In a word, these two established systems can provide reliable methods and reference value data for EMCV samples clinical detection.